Benzenediol lactones (BDLs) are fungal polyketide natural products with diverse biological activities. Different BDLs act as highly specific and potent inhibitors of the heat stress response system or diverse mitogen-activated protein kinases, while others are ligands for various receptors. Inhibition of MAP kinases translates to a potent antiproliferative activity against cancer cell lines that depend on mutant forms of these regulators. Similarly, the evolutionarily conserved chaperone Hsp90 is a validated target for cancer chemotherapy whose inhibition leads to a combinatorial blockade of multiple cancer-causing pathways. BDLs are biosynthesized by pairs of collaborating iterative polyketide synthases (iPKSs), representing a conceptual step towards the modular polyketide synthases. BDL biosynthesis also involves post-PKS modification of the polyketide scaffolds, catalyzed by specific tailoring enzymes. For the current application, we propose a four-pronged approach that will: 1. assemble an unprecedented genetic toolbox towards BDL biosynthesis; 2. use this toolbox to develop combinatorial biosynthetic methodologies to produce a large variety of unnatural BDL analogues and congeners; 3. decipher enzyme structural contributions to the biosynthetic rules of iPKSs; and 4. evaluate the produced BDLs in various biological assays to discover lead compounds for drug discovery. We will clone a large variety of BDL biosynthetic clusters from various fungi using traditional cloning pipelines as well as genome mining and synthetic biology. We will express the biosynthetic genes in a yeast heterologous host, and we will monitor the biosynthesis of new and known BDLs. We will investigate combinatorial biosynthesis by mixing and matching various iPKSs; creating hybrid synthases by domain swaps; and using post-PKS enzymes for combinatorial tailoring. We will also use domain exchanges, enzyme structural data, homology modeling and site-directed mutagenesis to define enzyme structural features that determine first ring cyclization geometry and chain termination repertoire. Finally, novel BDL congeners will be isolated, structurally identified, and evaluated for biological activities in various cell-based and in vitro biochemical assays. The lessons learned will advance our understanding of iterative enzymatic catalysis during BDL biosynthesis in particular and fungal polyketide biosynthesis in general. Improved methodologies for combinatorial biosynthesis will allow the production of novel BDLs and other polyketides. BDL analogs obtained in these experiments may provide lead compounds for developing treatments for cancer, immune system disorders, inflammatory or neurodegenerative conditions, and fungal infectious diseases.